Evaluation of the feeding safety of Moringa (Moringa oleifera L.) in the Sprague Dawley rat

This study aimed to evaluate the safety of Moringa by comparing the effects of different gavage doses of Moringa. The general behavior, body weight, food intake, blood indexes, serum biochemical indexes, and histopathology of rats were used to determine the safety threshold and to provide a reference for the further development and use of Moringa as animal feed. 40 Sprague Dawley rats were selected and given transoral gavage for 28 consecutive days. The T1, T2 and T3 groups were observed for general behavior, body weight, and food intake. Blood and serum biochemical indices were quantified, and histopathology was performed to evaluate the effect and safety of Moringa. The results of the toxicological test showed that (1) Only T1 groups experienced diarrhea. (2) The body weight and food intake of rats in each group were normal compared with the control group. (3) The hematological and serum biochemical indices of rats in the T1 group were significantly different from those of CK but were in the normal range; (4) The results of microscopic examination of the heart, liver, spleen, lung, and kidney of rats in each group were normal, but inflammation occurred in stomach and jejunum of rats in the T1 group, but not in the ileum. The gastrointestinal tract of rats in the T2 and T3 groups were normal. (5) No abnormal death occurred in any of the treatment groups.The results of this study revealed that gavage of Moringa homogenate at a dose of 6 g/kg BW can cause diarrhea in rats. Although there is no pathological effect on weight, food intake, blood and serum biochemical indicators in rats, there are pathological textures in the gastrointestinal tissue caused by diarrhea. Therefore, the safety threshold of Moringa homogenate should be ≤ 3 g/kg BW.


Sampling and pretreatment
Moringa was harvested from the Institute of Thermal Zone Ecological Agriculture, Yunnan Academy of Agricultural Sciences (Yunnan, China), in June 2021.The tested plants in this study are non endangered species, and the sample collection method is based on the principle of not damaging the ecological environment.
Homogenate preparation: 200 g of Moringa stems and leaves were weighed, added to 500 ml carboxymethylcellulose (CMC), and blended in a blender for 10 min.The suspension was used to make a high dose (400 mg/ ml; T1), a medium dose (200 mg/ml; T2), and a low dose (100 mg/ml; T3) gavage sample.Samples were used for a maximum of 4 days and were mixed evenly with ultrasonic homogenization before intragastric administration via oral gavage.CMC (0.5% aqueous solution) was administered to control animals (CK) by oral gavage.

Experimental animals
A total of 60 SD rats were purchased from Spelford (Beijing) Biotechnology Co., Ltd (Laboratory Animal Production License No. SCXK (Beijing) 2019-0010).SD rats were half male and half female, with a body weight of 239.3 ± 4.7.

Feeding environment and management
Animals were raised in a general animal laboratory environment with a temperature of 20-26 °C (daily temperature difference ≤ 4 °C) and a humidity of 40-70%.Animals were kept in a transparent rat cage 47 × 31.5 × 20 cm (length × width × height) and were fed SPF-grade rat growth breeding feed (Beijing KeoXieLi Feed Co, Ltd).The drinking water was homemade drinking water, which was autoclaved for the animals to drink freely.

Experimental treatments and toxicity assessment
Sixty SD rats were acclimatized for 7 days before gavage administration, during which clinical observation and recording were performed once daily.At the end of the acclimatization period, 40 rats were selected to enter the test after eliminating animals with abnormal conditions and too large or too small body weights.The animals were randomly divided into four groups of ten animals each (Table 1).
Rats were gavaged once a day, weighed twice a week, and fed rat growth breeding feed once a week for 28 days.Clinical observations were recorded once a day, including diet, behavioral activity, mental condition, fur condition, glandular secretion, respiration, fecal properties, local reactions, death, and symptoms of toxic reactions, including their onset, severity, duration, reversibility, and recovery time.At the end of the test period, blood and serum samples were collected from female rats for clinical tests.
At the end of the gavage period, the rats were killed and the organs of the rats in the high-dose group were observed first.If obvious histopathological changes or abnormalities were found in the high dose group, the relevant tissues or organs were examined in the medium and low dose groups.The heart, liver, spleen, lung, kidney, stomach and intestinal systems of the rats with lesions were removed and fixed in 10% neutral formalin fixative solution for more than 1 week, then sampling, sectioning, HE staining, full slide scanning and reading.

Statistical analysis
One-way ANOVA was performed using SPSS 17.0 statistical software for Windows.Multiple comparisons were performed using a Duncan multiple-range test when differences were significant.Results are expressed as mean ± standard deviation using p < 0.05 as the level of statistical significance.

Ethics approval and consent to participate
The animal management and experimental procedures of this study were conducted in accordance with the ARRIVE guidelines (PLoS Bio 8 (6), e10004122010) and approved by the Life Science Ethics Review Committee of Yunnan Agricultural University, with an ethics code of 202,209,005.In this study, according to the "Guidelines for euthanasia of experimental animals" (GB/T 39,760-2021), carbon dioxide was used to kill rats and every effort was made to minimize their pain.All methods were carried out in accordance with relevant guideline and regulations.

Mortality and general behavior
The mental condition of the rats in the fresh Moringa homogenate high-dose group was average during the gavage period, and the coat was rough (Table 2).However, the mobility and response were normal, and the rats showed slight diarrhea from day 1 to day 8 of gavage and severe diarrhea from day 9 to day 28 of gavage.The rats in the fresh Moringa homogenate medium and low-dose groups had good mental status, normal coat, mobility and reaction, and no diarrhea during the whole gavage period.

Body weight
The body weight of rats in all groups increased gradually with longer gavage time, and no abnormalities were observed (Table 3).The initial weight, final weight, and mean daily weight gain of the rats were not significantly different compared to the control group (P > 0.05), indicating no adverse effect of different doses of fresh Moringa on the weight gain.

Food intake
Food intake was calculated as the difference between the given amount of feed provided at the beginning of the test period and the amount of feed remaining at the end of one 4-day cycle.One value was recorded separately for each group per feed cycle.No significant difference (P > 0.05) was observed in the amount of food ingested by all four groups during the gavage period (Table 4).

Hematology and serum biochemistry
All hematological indices of all groups during the gavage period were within the normal physiological range (Table 5).The levels of platelet (PLT), mean platelet volume (MPV) and mean corpuscular hemoglobin www.nature.com/scientificreports/concentration (MCHC) in the blood of T1 rats were significantly lower than those of CK (P < 0.05).The blood indices of T2 and T3 rats were not significantly different from those of CK.
All the serum biochemical indices of all groups during the gavage period were within the normal physiological range (Table 6).Serum triglyceride (TG), blood glucose (GLU), creatine (Cr), and total cholesterol (CHOL) in T1 rats were significantly lower than those in CK (P < 0.05), but the difference with other treatment groups was not significant.Serum alkaline phosphatase (ALP) in T1 rats was significantly higher than those in CK, T2, and T3 (P < 0.05).Serum alanine aminotransferase (ALT) in T2 rats was significantly lower than that in CK (P < 0.05), and the difference with other treatment groups was not significant.Serum alanine aminotransferase (ALT) of T2 rats was significantly lower than that of CK (P < 0.05), but the difference with other treatment groups was not significant.Total bilirubin (TBIL) in the serum of T2 rats was significantly lower than that of CK (P < 0.05), but the difference with other treatment groups was not significant.

Histopathology
At the end of the gavage period, the stomach of rats in the CK group (Fig. 1) and the gastric mucosal layer of rats in the T1 group were damaged.The surface cell nuclei were deeply stained, and the interstitial space between cells was significantly increased, forming partial active proliferation (Fig. 2).Compared with the jejunum of rats  www.nature.com/scientificreports/ in the CK group (Fig. 3), the jejunum of rats in the T1 group showed no nuclear staining of superficial mucosal cells (Fig. 4), and a small number of superficial cells were shed compared with CK (Fig. 5), while the superficial cells were increased (Fig. 6) and enlarged compared to those in the jejunum of rats in the CK group (Fig. 7), forming a whole layer of cellular hyperplasia (Fig. 8).No abnormalities were seen in the ileum.

Discussion
In this study, a 28-day subacute toxicity test was conducted on SD rats using low, medium, and high doses (1.5 g/ kg, 3 g/kg, and 6 g/kg) of Moringa homogenate.The general behavioral condition, body weight, and food intake of rats in each group were recorded during the test period.Blood was collected at the end of the gavage period and analyzed for hematological and blood biochemical indices to evaluate the safety of Moringa.This experiment followed the "Chinese food 28-day oral toxicity test standard (GB15193.22-2014)".General behavioral status, body weight, and food intake are important indicators to characterize the dose-toxicity relationship of Moringa in rats [9][10][11][12] .Rats in the high-dose group of fresh Moringa homogenate showedrough coats, average mental status, normal movement and response, and diarrhea, while the medium and low-dose groups did not have diarrhea.This indicates that the oral administration of Moringa homogenate at 6 g/kg would have adverse effects on rats, while oral administration at 3 g/kg and below did not have any adverse effects on rats.The reason for diarrhea may be due to the high concentration in the high-dose group, which resulted in stretching of the stomach, behavioral adverse reactions, and mild diarrhea.It is also possible that the high dose of Moringa homogenate contained too high a concentration of a laxative component.
Body weight and ingestion status were more sensitive to the toxic effects of Moringa 13 .The body weight of rats in all groups tended to increase gradually with the duration of the gavage.Although the rats gavaged with high doses had diarrhea, the mean daily weight gain was not significantly different from that of the control group, demonstrating that high doses of fresh Moringa homogenate did not negatively affect the growth performance     www.nature.com/scientificreports/ of rats.There was no significant difference in the food intake of rats gavaged with fresh Moringa homogenate in all treatment groups compared with the control group.Blood plays an important role in maintaining normal metabolism and homeostasis in animals 14 .Any stimulus may affect the organism by causing changes in blood composition; therefore, blood tests are one of the most common and important tests in the diagnosis of animal diseases 15 .Animal hematology and blood biochemical indices are important and sensitive indicators of animal health and are essential in safety evaluation tests 16 .The hematological indices of the four groups of rats varied within the normal reference range.Platelet activity function is generally assessed by combining two parameters, PLT and MPV 17 .The blood PLT and MPV of rats in the fresh Moringa homogenate high-dose group were significantly lower than in the control group but still within the normal values and thus likely have no toxicological effect.High neutrophil levels are suggestive of inflammation, tissue damage, and pulmonary pathology 18 .The neutrophil levels in rats of all groups were within the normal range, indicating no toxicological effect.Red blood count (RBC), hemoglobin (HGB), and hematocrit (HCT) in the blood are important physiological indicators that respond to the characteristics of respiration, transport, and regulation of body fluid osmolality in animals 19 .No significant differences in RBC, HGB, and

Table 1 .
Grouping of toxicological tests on rats.

Table 2 .
General behavioral and diarrhea status of rats during the experimental period.

Table 3 .
Effect of Moringa on body weight of rats during the experimental period.The results were expressed as mean ± standard deviation and marked as significant level.

Table 4 .
Effect of Moringa on food intake of rats during the experimental period.The results were expressed as mean ± standard deviation and marked as significant level.

Table 5 .
Effect of Moringa on blood parameters of rats during the experimental period.The results are expressed as mean ± standard deviation.Different lowercase letters in the same parameter show significant differences (P < 0.05).WBC white blood cell count, NEU neutrophil count, LYM lymphocytes, MON monocytes, EOS eosinophil count, RBC red blood cell count, HGB hemoglobin, HCT hematocrit, MCV mean corpuscular volume, MCH mean corpuscular hemoglobin, MCHC mean corpuscular hemoglobin concentration, MPV mean platelet volume, PLT platelet.